Transposons produce genetic diversity during the life of individuals as well as over evolutionary time; their activities are implicated in many human genetic diseases. Maize is a model higher eukaryote ideally suited for analyzing the developmental regulation of transposons. The Mutator transposable element family is among the most active in higher eukaryotes; Mutator lines contain dozens of mobile Mu elements whose insertion elevates mutation frequency greater than 100-fold. Mutator transposon activities are stringently regulated during host development: Mu element excision, insertion and copy number amplification are all restricted to the terminal mitotic divisions of tissue differentiation. The outcome of Mutator activation depends on the tissue: in the soma, elements excise, but in germ cells elements amplify and insert (without excision, hence a very low germinal reversion frequency). The regulatory Mutator element was recently cloned and sequenced; it encodes two convergently transcribed genes with a shared termination region. With the "master" element in hand, our long-term objectives are to understand the developmental regulation of Mutator activities and how the interplay of host and element-encoded activities determines the differential behavior of Mu elements in somatic and germinal cells. Our current goals include characterizing Mu9, the presumptive "master" Mutator element, pursuing three specific aims: to use transient assays to characterize more completely the promoters, terminators and splicing of the two genes; to provide molecular proof that Mu9 is an "autonomous element" by assaying excision in transient assays and both excision and insertion in transgenic plants; and to determine the functions of the Mu9 gene products in transposition and/or element expression. Using a recently isolated Mutator line with novel phenotypes -- early somatic excision, high germinal reversion frequency, and drastic copy number reduction -- we will use RNase protection analyses to determine how the expression of Mu9 compares in standard and the variant Mutator lines. These data should provide insight into the developmental regulation of Mutator activity. Capitalizing on the drastic copy number reduction of the novel line, we will test a simplified gene tagging and cloning protocol.